The invention relates to an accurate determination of peptide concentrations in body fluids by mass spectrometry using internal reference substances. In medical research and medical diagnostics, protein concentrations in body fluids are usually determined by immuno assays, particularly by “Enzyme Linked Immuno-Sorbent Assays” (ELISA). Most often, because it is the safest method, a method called “Double Antibody Sandwich ELISA” is used, requiring two specific antibodies binding at two different epitopes of the protein molecule without sterical hindrance. The method is expensive, requires highly specific antibodies, does not allow for the application of internal references, and is not applicable for smaller peptides, because these rather tiny molecules in general do not offer two sufficiently separated specific binding epitopes for antibodies. Also, other types of immuno assays usually are not adaptable to these peptides.
Peptides—like the larger proteins—are biomolecules most essential for life. Blood and other body fluids contain many hundreds of physiologically active peptides: numerous peptide hormones, releasing hormones, neurotransmitters, and many others. Their concentration is highly informative about health or defects such as infections or other diseases; quite often small changes of the concentrations are decisive. A simple, safe, inexpensive, fast and highly precise analysis method, easily extensible to a multitude of different body fluid peptides, is urgently required. Occasionally, also peptide antibiotics and peptide toxins have to be analyzed quantitatively.
Diagnostic analysis procedures must fulfill strong quality standards. In most countries, permissions by state offices or official organizations are required to apply these methods in medical labs. In the USA, the FDA is responsible for the approval of medical analysis procedures. In some countries, state offices or official organizations must check the quality of a certain analytical procedure and the quality of the required devices according to rules which are specified by law.
In the field of bioorganic substances, mass spectrometry is most often directed to precise mass determinations for the identification of substances and only occasionally to quantitative analyses. Ionization methods for proteins and peptides, like electrospray ionization (ESI) or ionization by matrix assisted laser desorption (MALDI) were often regarded as non-quantitative. In fact, competitive processes during the ionization of mixtures of substances turn out to be advantageous for some substances and disadvantageous for others; so the degree of ionization depends on the mixture and the concentrations of the analyte substances. In the past, particularly MALDI was regarded as highly non-quantitative because early preparation methods of samples on mass spectrometric target plates (“dried droplets”) did not produce homogeneous samples, and the yield of ions varied over orders of magnitude from sample spot to sample spot.
More modern sample preparations use hydrophilic sample areas on hydrophobic MALDI target plates (U.S. Pat. No. 6,287,872 B1; GB 2 332 273 B; DE 197 54 978 C1; M. Schürenberg and J. Franzen, 1998), allowing for very homogeneous thin layer preparations. MALDI target plates made from electrically conductive plastics are commercially available, pre-prepared with thin layers of HCCA on 384 spots with 0.8 millimeter diameter (U.S. Pat. No. 6,825,465 B2; GB 2 391 066 B; DE 102 30 328 B4; M. Schürenberg, 2002). HCCA is an acronym for α-cyano-4-hydroxycinnamic acid, a widely used matrix substance for the ionization of proteins and peptides by matrix assisted laser desorption (MALDI). These thin layer preparations, in connection with suitable lasers, show excellent precision of quantitative analyses procedures with coefficients of variations (CV) in the order of five to ten percent, if suitable reference substances are used.
The best quantitative analysis procedures for measuring concentrations of analyte substances in fluids are based upon reference substances, measured in the same spectrum (internal standard). Most favorable reference substances are isotopically labeled substances of otherwise exactly the same type as the targeted analyte substance. As an example, a reference peptide for a target peptide may be a synthetically produced target peptide with just one arginine comprising six 13C and four 15N atoms instead of the native six 12C and four 14N atoms. This reference peptide is 10 dalton heavier than the target peptide. In a mass spectrum, the isotope groups of both substances are clearly separated. The reference substance is added in exactly known amounts to the fluid with the analyte substance, e.g., a blood plasma. Both analyte and reference substance are now extracted from the fluid, e.g., using magnetic beads with immobilized antibodies for the analyte substance. The extraction does not need to be extremely specific because any cross-reaction with other substances will be immediately visible in the mass spectrum, and usually does not disturb the analytical procedure. The extracted substances will then be removed from the extraction sites, e.g., immobilized antibody layers, and a sample preparation of the extracted substances will be prepared on a thin layer spot on a MALDI target plate. The ratio of peak heights or peak areas in the mass spectrum can be determined, and from this ratio, the concentration of the analyte substance can be calculated. Because the extraction process has exactly the same yield for both the analyte and the chemically identical reference, and the mass spectrometric sensitivity is also identical with respect to ionization and detection, the ratio reflects (in first order) the ratio of the concentrations.
Any quantitative mass spectrometric analysis procedure requires a safe recognition of the analyte by an accurate mass determination. We now change the subject from precise determination of peptide concentrations to accurate mass determination. In fact, mass spectrometers can only determine the ratio m/z of the ion mass m to the number z of unbalanced elementary charges of the ion. Where the terms “mass of an ion” or “ion mass” are used here for simplification, they always refer to this ratio m/z of the mass m to the dimensionless number of elementary charges z of the ion. This charge-related mass m/z has the physical dimension of a mass; it is often also called “mass-to-charge ratio”, although this is incorrect with regard to physical dimensions. In general, for the ionization of peptides by MALDI, z=1 is valid, in contrast to an ionization by electrospray (ESI), where most of the ions are multiply charged.
Any mass spectrometer delivers a series of ion current values forming a “spectrum”. The scale parameter x of the spectrum depends on the kind of mass spectrometer, it may be spatial position p, as in mass spectrometers with ion detection by photoplates or diode arrays, a scan voltage V, as in RF ion traps, or a flight time t in case of time-of-flight mass spectrometers. In general, the term “spectrum” acquired by a mass spectrometer should denote any of these ion current spectra noted above, but also any transformation of the ion current spectrum into other forms, as, for example, a spectrum of the frequencies f in Fourier-transform mass spectrometers, including even the final spectrum of the masses m.
For an accurate mass determination of ions by a given mass spectrometer, the mass spectrometer first has to be calibrated with a mixture of known calibration substances, covering the mass range of interest. This calibration procedure results in a function between the charge-related mass m/z of the ions and the scale parameter x along the spectrum, called a “calibration curve” m/z=f(x). Then a spectrum of the ions of an analyte substance can be measured and the masses m/z of the ions at a certain location x on the scale can be calculated using the calibration curve. For even more accurate mass measurements, one adds to the analyte substance one or more known mass reference substances and corrects the masses of the analyte substance using the mass differences, found for the mass reference substances between calculated and true masses (method with “internal mass reference”).
The basis of all these calibration methods is always the exact determination of the precise scale parameter x for an individual peak on the spectrum scale axis. Any spectrum acquired by a mass spectrometer consists of a large series of individual digital values, either measurement values or transformed measurement values, where the indices of the series represent the spectrum scale. For exact mass determinations, one must derive the accurate position, frequency, or time value from a measured (spatial or temporal) profile of the measured ion current values across a peak. Usually, the ion current peak consists of four to ten measurement values above background noise. In the simplest case a centroid formation of the individual measured values is used. In more elaborate but more accurate methods a theoretically derived or experimentally determined function is fitted into the measured profile of a mass peak, from which the optimal positions, frequencies or time values of the peaks are derived.
In the following, we shall restrict the description for reasons of clarity to time-of-flight spectra only; this should, however, not mean that the invention will be restricted to this type of spectra. In modern time-of-flight mass spectrometers (TOF-MS), single spectra are obtained in 50 to 200 microseconds, using measuring rates up to 4 giga-samples per second and more. The acquisition of single spectra is repeated 5,000 to 10,000 times per second; the series of 200,000 to 800,000 measuring values are added in real time, value for value over a predetermined time period to yield a sum spectrum in which the signal-to-noise ratio is greatly improved and the dynamic measuring range is enlarged. The determination of the exact flight time of the ions of a peak is then performed using the series of summed measuring values of this sum spectrum. The flight time determination of a peak constitutes the main source of inaccuracies of the mass determination.
For more complex substances, as for biopolymers like peptides, measured with sufficient mass resolutions R, the spectrum of the molecular ions always shows several peaks with ions of the same elemental but different isotopic composition, the peaks being one atomic mass unit apart from each other. These peaks are called here an “isotope group”. FIGS. 1 to 3, which show ion current peaks versus a scale factor x, present three examples for peptides with masses of 1,000, 2,000 and 3,000 dalton, respectively. The lightest ions form the “mono-isotopic peak” of the group, composed only of 1H, 12C, 14N, 16O, 31P and 32S. There are methods to calculate the exact form of the isotope group of peaks from the elemental composition and the known isotope abundances of the elements, but these calculations are rather complicated and slow.
In document U.S. Pat. No. 6,188,064 B1 (GB 2 333 893 B; DE 198 03 309 01; C. Koester, 1998), a highly precise method is described for the determination of the flight time of the mono-isotopic peak, using a process of fitting a whole family of superimposed bell-shaped curves of known mass distances and averaged peak height relations into the measured signal pattern of the complete isotope peak group, instead of fitting just one single bell-shaped curve into a measured signal profile of a single ion peak. This method has become widely known by the name “SNAP”. The method is directed to an exact determination of the masses of ions of unknown substances for their identification. While the substances themselves are unknown, the class of substances, e.g., proteins, is generally known. Therefore, the peak height distribution of the isotope group is calculated as an average for the class of substances, using an average of the elemental composition of that class and the known isotope abundances of the elements. As can be seen from FIGS. 1 to 3, the average peak height distribution is strongly dependent on the mass of the ions. Therefore, a series of averaged peak height distributions is calculated once for ions of different masses of this class of substances, stored in a table, and used over and over, calculating the averaged peak height distribution at a given mass by interpolation from the stored distributions in the table. This procedure saves time, because the calculation of peak height distributions from averaged elemental composition and isotope abundances is rather complicated and cannot be performed for each isotope group in a complicated spectrum in an acceptable time span.
Coming back to the determination of peptide concentrations, to improve the precision of the analysis procedure, the readily available SNAP procedure was applied to the isotope groups of target and reference peptide. Regrettably, however, no improvement of the precision could be observed.
A highly precise procedure is still sought for the determination of the ratio of the target peptide ion current to the reference peptide ion current, represented by the detectable peaks in the measured spectrum and used for the quantitative analysis of peptides in fluids. Because the analytical procedure should also be used for diagnostic purposes, it is a most essential requirement to enable the procedure to recognize, by built-in quality indicators, any technical faults and disturbances in each individual analysis.